Colorectal cancer is the second leading cause of cancer deaths in the United States but is preventable through the removal of precancerous adenomatous polyps. New vessel growth (angiogenesis) is a prerequisite for polyp growth beyond 2-3mm3 and is consequently an attractive target for cancer prevention. Endothelial cells are sensitive to killing by epithelial-derived membrane-bound Fas ligand (FasL) but are resistant to killing by cleaved FasL in vitro. Thus, in epithelial tissues that express FasL, such as precancerous colorectal polyps, cleavage of FasL may be required before angiogenesis and tumor progression can proceed. We propose that angiogenesis enabled by FasL cleavage or its loss of expression is an important checkpoint for the development of colorectal cancers. In support of this hypothesis, our preliminary data suggest that: 1) colorectal tumor angiogenesis and tumor growth are stimulated when functional FasL protein is absent; 2) FasL expressed in colorectal cancer cells is cleaved to a non-functional cleaved form by the matrix metalloproteinase, matrilysin and; 3) FasL gene expression is silenced by transforming growth factor beta (TGFbeta) signaling proteins Smad2 and Smad4. These observations provide the foundation of our CENTRAL HYPOTHESIS that inhibition of tumor angiogenesis by epithelial-derived membrane-bound FasL is controlled at the level of FasL protein cleavage or gene silencing. This hypothesis will be tested through the following specific aims: SPECIFIC AIM 1: Demonstrate that epithelial-derived FasL and cleaved FasL differentially regulate endothelial cell apoptosis and tumor growth and tumor angiogenesis in vitro and in vivo; SPECIFIC AIM 2: Demonstrate that cleavage of FasL by matrilysin is required for angiogenesis in colorectal tumors expressing FasL; and SPECIFIC AIM 3: Determine the mechanism through which TGFbeta signaling proteins negatively regulate beta-catenin/Tcf transactivation through the Tcf response element. We are optimistic that successful completion of this proposal will provide new insights into how epithelial-endothelial cells interact to modulate colorectal tumor growth and angiogenesis. In addition, these studies will provide further insight into FasL regulation at both the gene and protein level. These insights may lead to novel and effective therapeutic strategies that target this pathway to prevent angiogenesis, and thereby prevent colorectal tumor development growth and spread.